Topical Brimonidine Tartrate Ophthalmic Solution

ABSTRACT

The present invention provides an aqueous ophthalmic composition comprising an alpha-2 adrenergic receptor agonist and a non-ionic cellulosic polymer, the solution having a pH less than 6.5. The present invention also provides an aqueous ophthalmic composition comprising an alpha-2 adrenergic receptor agonist and a benzododecinium halide. Also provided are methods of manufacture, use and method of reducing intraocular pressure in the patient in need thereof.

FIELD OF THE INVENTION

The present invention provides an aqueous ophthalmic compositioncomprising an alpha-2 adrenergic receptor agonist and a non-ioniccellulosic polymer, the solution having a pH less than 6.5. The presentinvention also provides an aqueous ophthalmic composition comprising analpha-2 adrenergic receptor agonist and a benzododecinium halide. Alsoprovided are methods of manufacture, use and method of reducingintraocular pressure in a patient in need thereof.

BACKGROUND OF THE INVENTION

The present invention provides a topical ophthalmic solution comprisingan alpha-2 adrenergic receptor agonist such as brimonidine tartrateand/or pharmaceutically acceptable excipients wherein the ophthalmicsolution is soluble enough to achieve therapeutic efficacy is soluble ata lower pH range of from about 5.5 to about 6.5.

The ophthalmic solution of the present invention contains from about0.05% to about 0.2% (w/v) of brimonidine tartrate.

Brimonidine tartrate is an alpha-2-adrenergic agonist that reduces theelevated intraocular pressure (IOP) of the eye that is associated withglaucoma. The topical use of brimonidine to lower intraocular pressurein patients with glaucoma or ocular hypertension is known.

The first ophthalmic brimonidine product in the U.S. was approved by theFDA in 1996. That product, sold under the trade name Alphagan®,contained brimonidine in the form of brimonidine tartrate at aconcentration of 0.2%. According to the product label, Alphagan® isadjusted (with NaOH or HCl) to a pH between 5.6 and 6.6, and furthercontains citric acid, polyvinyl alcohol, sodium chloride, sodiumcitrate, and purified water. The preservative contained in Alphagan® isbenzalkonium chloride, the most widely used preservative for topicalophthalmic compositions.

In 2001, a second ophthalmic brimonidine product was approved by theU.S. FDA. This product, sold under the trade name Alphagan® P, containedbrimonidine tartrate at two brimonidine concentrations, 0.15% and 0.1%,each of which is lower than the 0.2% brimonidine concentration inAlphagan®. Alphagan® P has a pH between 7.15 and 7.8, a range that ishigher than that of Alphagan®. According to the product label, the lowerconcentration Alphagan® P formulation is sold at a pH of 7.4 to 8.0; thehigher concentration is sold at a pH of 6.6 to 7.4. The preservativecontained in Alphagan® P is chlorine dioxide. See U.S. Pat. Nos.5,424,078 and 6,562,873. Alphagan® P also contains an anionic solubilityenhancing component (carboxymethylcellulose) to help solubilize thebrimonidine that is unionized at the pH of the compositions. Currentlythe innovator has discontinued marketing Alphagan®, the higherconcentration, and low pH product.

Brimonidine has a pKa of 7.4. Hence, at pH below 6.6, it will besubstantially ionized. For example, at a pH of 6.4, brimonidine is about90% ionized. It is well known that ionized ophthalmic drugs have greatlyreduced ocular permeability. It would have been expected that at a pHbelow 6.6 or 6.5, brimonidine would not permeate ocular tissue well,thereby reducing its efficacy compared to a higher pH product. It isbelieved that this is why the drug concentration in Alphagan® issubstantially higher than in the higher-pH product Alphagan® P.

To overcome this disadvantage, the inventors of the present inventionhave formulated the current product with a non-ionic cellulosederivative, preferably hydroxypropyl methylcellulose (e.g., HPMC E4Mgrade). Without being bound by theory, it is believed that such apolymer reduces the surface tension to around 45 dynes/cm from about 72dynes/cm, thereby helping to spread the drop more effectively around theocular surface. Moreover, due to its viscosity, it is believed toincrease retention of the drop in eye. Both of these effects arebelieved to help increase penetration of the drug. Surprisingly theefficacy of this invention appears to be similar to that of Alphagan® P,if not better (statistically significant), in spite of the presentinventive compositions being at lower pH, wherein most of the drug is inionized state.

The inventors of the present invention have formulated an ophthalmicsolution wherein the pH of the formulation is acidic, preferably havingpH from about 5.5 to about 6.5, yet is surprisingly effective comparedto other low-pH products (e.g., Alphagan®). The lower pH also removesany need to use a solubility enhancing agent(s) because brimonidine ishighly soluble at acidic pH (5.5-6.5) (though a solubility enhancingagent may be used if desired).

Further, the inventors of the present invention have surprisinglyimproved the stability of brimonidine significantly over the commercialproduct (Alphagan® P). The data are shown in Table 6.

Brimonidine tartrate has highly pH-dependent solubility. The solubilitydecreases sharply as the pH increases as shown in Table 1 (Refer U.S.Pat. No. 6,627,210 B2 page no. 13-14).

TABLE 1 Solubility of brimonidine tartrate a solubility enhancing agentover pH range of 5 to 8. Study 1 Study 2 pH Solubility pH Solubility5.55 ≥164.4 5.5 ≥200.6 5.92 132.6 5.92 160.8 6.14 30.4 6.06 50.1 6.577.55 6.9 3.19 7 2.69 7.4 1.19 7.45 1.17 7.77 0.63 7.83 0.62 7.86 0.587.88 0.54

The original Alphagan® included a detergent preservative, benzalkoniumchloride, which was known to be somewhat irritating to the eye.

Gasset and Grant et al. showed that BAC accumulates in ocular tissue andremains there for long periods, adversely affecting both the cornealsurface and the conjunctiva. Therefore, cessation of the medications maynot immediately improve the condition and function of the ocularsurface. These findings also suggest that corneal cell necrosis mayoccur in some patients who are taking multiple BAC-preserved ocularmedications over long periods of time, even when the amount of BAC inany one medication is below the threshold concentration at whichnecrosis occurs.

It is well known in the reference literature that small organiccompounds, such as benzalkonium chloride (BAC), chlorhexidine,thimerosal have excellent antimicrobial activity; however, it is nowknown that these small organic antimicrobials are often toxic to thesensitive tissues of the eye and can accumulate in cornea, contactlenses, particularly soft, hydrophilic contact lenses. Medications withBAC may cause disruption of the corneal surface with lowerconcentrations of BAC.

The preservative in Alphagan® P is stabilized chlorine dioxide (“SCD”),an oxidative preservative that was known to be compatible with the eyebut Chlorine dioxide is not an ideal preservative ingredient. It is anoxidative preservative and it would oxidize brimonidine. It is difficultto stabilize and is light-sensitive as referred in U.S. Pat. No.7,265,117.

It has been unexpectedly found that benzododecinium bromide is aquaternary ammonium compound that does not form a precipitation withbrimonidine at pH of around 6.0. Benzododecinium bromide forms an ionpair with brimonidine, thereby neutralizing the charge of brimonidine.Thus it was surprising to find that benzododecinium bromide is aquaternary ammonium compound that does not form precipitation withbrimonidine. In the same conditions brimonidine forms a hazy solutionwith benzalkonium chloride.

Hence there is an unmet medical need to prepare a topical ophthalmicsolution comprising an alpha-2 adrenergic receptor agonist such asbrimonidine tartrate and/or pharmaceutically acceptable excipientswherein it does not comprises a solubility enhancing agent and anoxidative preservative. Instead, when the preservative is included inthe said solution, the preservative is preferably benzododeciniumbromide. Benzododecinium bromide is an effective preservative foralpha-2 adrenergic receptor agonists (e.g., brimonidine) in acidicconditions.

It was surprisingly found by the inventors of the present invention thatthe formulation of the present invention has shown statisticallysignificant IOP lowering efficacy when administered to normotensive andwater loaded New Zealand white rabbits by ocular route. The onset ofstatistically significant IOP lowering efficacy was 15 minutes earlierin present invention which is same as compared to Alphagan® P, whereasboth the formulations were found to be comparable at each time pointobserved, which indicates that the IOP lowering efficacy of both testformulations (Alphagan® P and the present invention) were statisticallycomparable.

OBJECT OF THE INVENTION

The main object of the present invention is to develop an aqueousophthalmic composition comprising an alpha-2 adrenergic receptor agonistand a non-ionic cellulosic polymer and devoid of anionic cellulosicpolymer, the solution having a pH less than 6.5.

Another object of the present invention is to develop an aqueousophthalmic composition of brimonidine compatible with quaternaryammonium compound used as a preservative other than oxidativepreservatives without forming precipitate.

Yet another object of the present invention is to develop a method toprepare an aqueous ophthalmic composition comprising an alpha-2adrenergic receptor agonist and a non-ionic cellulosic polymer anddevoid of anionic cellulosic polymer, wherein the solution having a pHless than 6.5.

Yet another object of the present invention provides a method forreducing intraocular pressure in a patient in need thereof comprisingadministering an aqueous ophthalmic composition comprising an alpha-2adrenergic receptor agonist and a non-ionic cellulosic polymer, whereinthe solution having a pH less than 6.5.

SUMMARY OF THE INVENTION

The present invention provides an aqueous ophthalmic compositioncomprising an alpha-2 adrenergic receptor agonist and a non-ioniccellulosic polymer, the solution having a pH less than 6.5. The presentinvention also provides an aqueous ophthalmic composition comprising analpha-2 adrenergic receptor agonist and a benzododecinium halide. Thepresent invention also provides a method of reducing intraocularpressure in a patient in need thereof, comprising administering to thepatient the composition of the present invention, the administeredcomposition comprising an effective amount of the alpha-2 adrenergicreceptor agonist.

The alpha-2 adrenergic receptor agonist preferably comprises brimonidineor a pharmaceutically acceptable salt thereof, preferably brimonidinetartrate. The amount of brimonidine or pharmaceutically acceptable saltthereof is preferably 0.05-0.2% (w/v) brimonidine tartrate, based onbrimonidine free base. The alpha-2 adrenergic receptor agonist isdissolved or suspended in the aqueous composition, preferably dissolved.

The non-ionic cellulosic polymer preferably comprises hydroxypropylmethylcellulose, preferably 0.05-1.5% (w/v) hydroxypropylmethylcellulose.

The pH of the composition is preferably less than or up to 6.6. The pHof the composition is preferably at least or greater than 5.5.

The composition preferably comprises a preservative, preferablybenzododecinium halide, more preferably benzododecinium bromide. Whenthe preservative is present, the composition preferably comprises 0.005%to 0.03% (w/v) benzododecinium halide, preferably benzododeciniumbromide.

The composition preferably does not comprise an anionic solubilityenhancing component, and preferably does not comprise carboxymethylcellulose. The composition preferably does not comprise an oxidativepreservative, and preferably does not comprise an oxy-chloropreservative.

Surprisingly the formulation of the present invention is more stablethan the commercial product, Alphagan® P.

The present invention provides a topical ophthalmic solution comprisingan alpha-2 adrenergic receptor agonist such as brimonidine tartrateand/or pharmaceutically acceptable excipients wherein the ophthalmicsolution is soluble enough to achieve therapeutic efficacy at an acidicpH. Preferably, the acidic pH is less than 7, more preferably less thanor about 6.7 or 6.5, more preferably less than or about 6.6 or 6.5.Other than physiological considerations (e.g., eye irritation), there isno particular lower limit on the pH. Preferably, the pH is greater thanor about 5, more preferably greater than or about 5.25, or 5.5.

More particularly the present invention provides a topical ophthalmicsolution comprising an alpha-2 adrenergic receptor agonist such asbrimonidine tartrate and/or pharmaceutically acceptable excipients whichdoes not comprises a solubility enhancing agent and an oxidativepreservative. When the preservative is included in the said solution,the preservative is quaternary ammonium compound, preferablybenzododecinium bromide.

More preferably, benzododecinium bromide is the only quaternary ammoniumcompound which is included in said solution.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIG. I shows the intraocular pressure (IOP) lowering effect of Group I(Alphagan® P) and Group II (present invention) on normotensive NewZealand White Rabbits for treated left eye. The other (right) eye isleft untreated and serves as control.

FIG. II shows the intraocular pressure (IOP) lowering effect of Group I(Alphagan® P) and Group II (present invention) on New Zealand WhiteRabbits for treated left eye in water loaded rabbits. The other (right)eye is left untreated and serves as control.

FIG. III shows the total Impurity levels in various brimonidineformulations i.e. commercial (Alphagan® P) and present inventionformulation (brimonidine tartarate 0.1%) at 40° C./NMT 25% RH.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “BAC” wherever appears is an abbreviation for“benzalkonium chloride”.

As used herein, the “SCD” wherever appears is an abbreviation for“stabilized chlorine dioxide”.

As used herein, the “NMT” wherever appears is an abbreviation for “notmore than”.

As used herein, the “RH” wherever appears is an abbreviation for“relative humidity”.

As used herein, the “HPMC” wherever appears is an abbreviation for“hydroxypropyl methyl cellulose”.

As used herein, the “IOP” wherever appears is an abbreviation for“intraocular pressure”.

Unless indicated otherwise, all ingredient amounts are presented inunits of % weight/volume (% w/v).

Brimonidine tartrate is a known compound that can be made by knownmethods and is commercially available. See, for example, German PatentNo. 2,538,620.

In one embodiment of the present invention, an aqueous ophthalmiccomposition comprising an alpha-2 adrenergic receptor agonist and anon-ionic cellulosic polymer.

In another embodiment of the present invention, an aqueous ophthalmiccomposition comprising an alpha-2 adrenergic receptor agonist, anon-ionic cellulosic polymer and devoid of anionic cellulosic polymer.

In yet another embodiment of the present invention, an aqueousophthalmic composition comprising an alpha-2 adrenergic receptoragonist, a non-ionic cellulosic polymer and devoid of anionic cellulosicpolymer and oxidative preservative.

In yet another embodiment of the present invention, an aqueousophthalmic composition comprising an alpha-2 adrenergic receptoragonist, a non-ionic cellulosic polymer and devoid of anionic cellulosicpolymer and oxidative preservative, optionally along with apreservative.

In yet another embodiment of the present invention, an aqueousophthalmic composition comprising an alpha-2 adrenergic receptor agonistand a preservative.

In yet another embodiment of the present invention, an aqueousophthalmic composition comprising an alpha-2 adrenergic receptor agonistand a preservative and devoid of an anionic solubility enhancingcomponent.

In yet another embodiment of the present invention, an aqueousophthalmic composition comprising an alpha-2 adrenergic receptor agonistand a preservative and devoid of an anionic solubility enhancingcomponent and oxidative preservative.

In yet another embodiment of the present invention, an aqueousophthalmic composition comprising an alpha-2 adrenergic receptor agonistand a preservative and devoid of an anionic solubility enhancingcomponent and oxidative preservative, optionally along with a non-ioniccellulosic polymer.

According to the present invention, an alpha-2 adrenergic receptor isselected from Brimonidine or a pharmaceutically acceptable salt orsolvate or hydrate thereof.

According to the present invention, a non-ionic cellulosic polymer isselected from hydroxypropyl methylcellulose, hydroxypropyl cellulose,hydroxymethyl cellulose, hydroxyethyl cellulose or combinations thereof.More preferable is hydroxypropyl methylcellulose.

According to the present invention, a preservative is selected frombenzododecinium halide, chlorobutanol, sodium perborate, cetrimoniumchloride, thiomersal, methyl parahydroxybenzoate, propylparahydroxybenzoate, sorbic acid and derivatives thereof, polyquaterniumammonium chloride, polyaminopropyl biguanide, phenyl mercuric nitrate,phenyl mercuric acetate, hydrogen peroxide. More preferably isbenzododecinium halide. Still more preferable is benzododeciniumbromide.

In yet embodiment of the present invention, an aqueous ophthalmiccomposition comprising a brimonidine in the range of 0.01-0.5 (w/v),more preferable in the range of 0.05-0.2% (w/v) and a non-ioniccellulosic polymer in the range of 0.05-1.5% (w/v), more preferable inthe range of 0.1-1.0% (w/v).

In yet embodiment of the present invention, an aqueous ophthalmiccomposition comprising a brimonidine in the range of 0.01-0.5 (w/v),more preferable in the range of 0.05-0.2% (w/v) and non-ionic cellulosicpolymer in the range of 0.05-1.5% (w/v), more preferable in the range of0.1-1.0% (w/v) along with a preservative in the range of 0.001% to 0.1%(w/v) and more preferable 0.005% to 0.03% (w/v).

The ophthalmic solution of the present invention contains an effectiveamount of alpha-2 adrenergic agonist, preferably brimonidine. Preferredamounts are at least 0.01%, more preferably at least about 0.02%, morepreferably at least about 0.05%, percentages being w/v. Preferred amountare less than about 1.0%, more preferably less than about 0.5%, 0.2%.Some preferred amounts include 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, and0.15%. Preferred ranges include ranges formed from any two of theseamounts. Weight of brimonidine is based on the free base. Salts ofbrimonidine are included, preferably including brimonidine tartrate.

In an embodiment the present invention provides a topical ophthalmicsolution comprising an alpha-2 adrenergic receptor agonist such asbrimonidine tartrate and/or pharmaceutically acceptable excipientswherein the ophthalmic solution is at an acidic pH.

In another embodiment the present invention provides a topicalophthalmic solution comprising an alpha-2 adrenergic receptor agonistsuch as brimonidine tartrate and/or pharmaceutically acceptableexcipients which does not comprises a solubility enhancing agent and anoxidative preservative. Instead, when the preservative is included inthe said solution, the preservative is a quaternary ammonium compound,preferably benzododecinium halide.

In yet another embodiment benzododecinium halide, preferablybenzododecinium bromide is the only quaternary ammonium compound whichis included in said solution.

It has been found that benzododecinium halide is a quaternary ammoniumcompound that does not form a precipitation with brimonidine at pH ofaround 6.0. Benzododecinium halide (e.g., bromide) forms an ion pairwith brimonidine, thereby neutralizing the charge of brimonidine. It hadbeen believed that quaternary ammonium preservatives other that BAC weregenerally not compatible with brimonidine. Thus it was surprising tofind that benzododecinium halide is compatible with brimonidine. Inparticular, benzododecinium bromide does not form precipitation withbrimonidine. The observation that benzododecinium bromide is compatiblewith brimonidine indicates it should be an effective preservative forvarious dosage forms comprising brimonidine, including solutions andsuspensions.

Multi-use containers preferably comprise a preservative. Single-usecontainers may optionally comprise preservative. When used, any safe andeffective preservative amount of benzododecinium halide, preferablybromide, may be used in compositions of the present invention. Amountsbetween 0.001% to 0.1% w/v are suitable. An amount of about 0.005% to0.03% is preferred. A preferred composition comprises about 0.01% (e.g.,0.012%).

As a result of the various studies, it has been found that there is noneed to add any solubility enhancing agent because brimonidine issoluble at acidic pH range between about 5.5 to about 6.5.

Compositions of the present invention preferably comprise a non-ioniccellulosic polymer. The non-ionic cellulosic polymer can be anywater-soluble polymer that increases the viscosity of the composition.Non-limiting examples include hydroxypropyl methyl cellulose (HPMC),hydroxypropyl cellulose (HPC), hydroxyethyl cellulose, hydroxymethylcellulose and other modified celluloses. A preferred non-ioniccellulosic polymer is HPMC. Any amount of non-ionic cellulosic polymermay be used to obtain a suitable viscosity. Preferred amounts include atleast 0.05%, more preferably at least 0.1% or 0.2%, all percentagesbeing w/v. Preferred amounts include up to 1.5%, more preferably up to1%. Some preferred amounts include 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%and 0.9%. Preferred ranges include ranges formed from any two of theseamounts. For the purposes of the present invention, the non-ioniccellulosic polymer does not include carboxymethylcellulose.

Surprisingly the formulation of the present invention is more stablethan the commercial product, Alphagan® P. The inventors of the presentinvention have further studied extensively and completed the presentinvention. Namely, the present invention relates to:

1. An aqueous ophthalmic composition comprising an alpha-2 adrenergicreceptor agonist, a non-ionic cellulosic polymer and devoid of anioniccellulosic polymer, optionally along with a preservative andpharmaceutically acceptable excipients, wherein the pH of saidcomposition is less than 6.5.

2. The composition according to the above 1 wherein the alpha-2adrenergic receptor agonist is brimonidine or a pharmaceuticallyacceptable salt or solvate or hydrate thereof. 3. The compositionaccording to the above 1 or 2, wherein the composition comprises0.01-0.5% (w/v) of brimonidine tartrate.

4. The composition according to the above 1 or 2, wherein thecomposition comprises 0.05-0.2% (w/v) of brimonidine tartrate.

5. The composition according to the above 1, wherein the non-ioniccellulosic polymer is selected from the group consist of hydroxypropylmethylcellulose, hydroxypropyl cellulose, hydroxymethyl cellulose,hydroxyethyl cellulose or combinations thereof.

6. The composition according to the above 1 or 5, wherein the non-ioniccellulosic polymer is hydroxypropyl methylcellulose.

7. The composition according to the above 6, wherein the compositioncomprises of 0.05-1.5% (w/v) hydroxypropyl methylcellulose.

8. The composition according to the above 7, wherein the compositioncomprises of 0.1-1.0% (w/v) hydroxypropyl methylcellulose.

9. The composition according to the above 1 to 8, wherein the pH is lessthan 6.5.

10. The composition as claimed in claims 1 to 8, wherein the pH of thecomposition is in the range of 5.5-6.5.

11. The composition according to the above 1, wherein the preservativeis selected from the group consist of benzododecinium halide,chlorobutanol, sodium perborate, cetrimonium chloride, thiomersal,methyl parahydroxybenzoate, propyl parahydroxybenzoate, sorbic acid andderivatives thereof, polyquaternium ammonium chloride, polyaminopropylbiguanide, phenyl mercuric nitrate, phenyl mercuric acetate, hydrogenperoxide.

12. The composition according to the above 1 or 11, wherein thepreservative is benzododecinium halide.

13. The composition according to the above 1 or 11, wherein thepreservative is benzododecinium bromide.

14. The composition according to the above 1 to 13, wherein thecomposition is further devoid of an oxidative preservative.

15. A method of reducing intraocular pressure in a patient in needthereof, comprising administering to the patient the compositionaccording to the above 1 to 14, the administered composition comprisingan effective amount of the alpha-2 adrenergic receptor agonist.

16. An aqueous ophthalmic composition comprising an alpha-2 adrenergicreceptor agonist and benzododecinium halide.

17. The composition according to the above 16, wherein the compositioncomprises 0.001% to 0.1% (w/v) of benzododecinium halide.

18. The composition according to the above 17, wherein the compositioncomprises 0.005% to 0.03% (w/v) of benzododecinium halide.

19. The composition according to the above 16 to 18, wherein thebenzododecinium halide is benzododecinium bromide.

20. The composition according to the above 16, wherein the alpha-2adrenergic receptor agonist is brimonidine or a pharmaceuticallyacceptable salt or solvate or hydrate thereof.

21. The composition according to the above 16, wherein the compositionfurther comprises a non-ionic cellulosic polymer.

22. The composition according to the above 21, wherein the non-ioniccellulosic polymer is selected from the group consist of hydroxypropylmethylcellulose, hydroxypropyl cellulose, hydroxymethyl cellulose,hydroxyethyl cellulose and combinations thereof.

23. The composition according to the above 21 or 22, wherein thenon-ionic cellulosic polymer is hydroxypropyl methylcellulose.

24. The composition according to the above 16 to 23, wherein the pH ofthe composition is in the range of 5.5-6.5.

25. The composition according to the above 16 to 24, wherein thecomposition is devoid of an anionic solubility enhancing component.

26. The composition according to the above 25, wherein the compositionis further devoid of an oxidative preservative.

27. An aqueous ophthalmic composition comprising

-   -   a) brimonidine tartrate in an amount of 0.01-0.5% (w/v);    -   b) hydroxypropyl methylcellulose in an amount of 0.1-1.0% (w/v)    -   c) benzododecinium halide in an amount of 0.001% to 0.1% (w/v)        which is devoid of anionic cellulosic polymer, wherein the pH of        said composition is less than 6.5.

28. A method of reducing intraocular pressure in a patient in needthereof, comprising administering to the patient the compositionaccording to the above 16 to 27, the administered composition comprisingan effective amount of the alpha-2 adrenergic receptor agonist.

29. Use of a composition according to the above 1 to 14 and 16 to 27 forreducing intraocular pressure in a patient in need thereof comprisingsafe and effective amount of alpha-2 adrenergic receptor agonist.

30. A method for the preparation of an aqueous ophthalmic compositionaccording to the above 1 to 14 and 16 to 27, wherein the methodcomprises of:

a) Adding required quantity of non-ionic cellulosic polymer to one partof water for injection at 60-70 ° C. under stirring to form thesolution.

b) Cooling the solution as obtained in step a) to room temperature understirring to form Part-A of the solution.

c) Adding pharmaceutically acceptable excipients as described herein,preservative and alpha-2 adrenergic receptor agonist under stirring toanother part of water for injection which is cooled to room temperaturebefore such addition to form Part-B of the solution.

d) Adding Part-B solution to Part-A solution under stirring to form asolution.

e) Checking and adjusting the pH of the solution as obtained in step d)with 1N HCl/1N NaOH and finally making up the volume to 100% with waterfor injection to obtain an aqueous ophthalmic composition.

In addition to brimonidine or salt thereof, the solution of the presentinvention preferably also contains buffer components to stabilize ormaintain the ophthalmic formulation at the desired pH. Any suitablebuffer component can be employed which is compatible with the otheringredients of the ophthalmic solution, and which does not havedeleterious or toxic properties which could harm the eye. Examples ofsuitable ophthalmically acceptable buffer components include acetatebuffers, citrate buffers, phosphate buffers, borate buffers and mixturesthereof. Specific buffer components useful in the present inventioninclude boric acid, sodium borate, sodium phosphates, including mono,di- and tri-basic phosphates, such as sodium phosphate monobasicmonohydrate and sodium phosphate dibasic heptahydrate, and mixturesthereof.

In an embodiment of the present invention, tonicity adjusting agents maybe added and included without limitation such as glycerin, sorbitol,sodium hydroxide, sodium chloride, potassium chloride, and mannitol,dextrose, propylene glycol and combinations thereof or any othersuitable ophthalmically acceptable tonicity adjusting agents.

In one embodiment, the tonicity component is selected from inorganicsalts and mixtures thereof.

The amount of ophthalmically acceptable tonicity component utilized canvary widely. In one embodiment, the tonicity component is preferablypresent in the ophthalmic formulation in an amount in the range of about0.5 to about 0.9 weight/volume percent of the formulation.

Typical of ophthalmically acceptable inorganic salt tonicity componentsare alkali metal chlorides and alkaline earth metal chlorides, such assodium chloride, potassium chloride, calcium chloride and magnesiumchloride.

The pH adjusting agents include hydrochloric acid, sodium hydroxide,phosphoric acid, acetic acid and the like.

In another embodiment one or more additional components can be includedin the present solution based on the particular application for whichthe formulations are made. The additional component or componentsincluded in the present solution are chosen to impart or provide atleast one beneficial or desired property to the solutions. Examples ofsuch additional components include cleaning agents, non-ionic polymers,nutrient agents, sequestering agents, viscosity builders, contact lensconditioning agents, antioxidants, and the like.

These additional components are each included in the present ophthalmicsolution in an amount effective to impart or provide the beneficial ordesired property to the compositions.

Exemplary non-ionic polymers include, but are not limited to, Povidone(PVP: polyvinyl pyrrolidone), polyvinyl alcohol, copolymer of PVP andpolyvinyl acetate, gelatin, polyethylene oxide, acacia, dextrin, starch,polyhydroxyethylmethacrylate (PHEMA), water soluble nonionicpolymethacrylates and their copolymers, modified non-cellulosicpolysaccharides, nonionic gums, nonionic polysaccharides, and/ormixtures thereof, such as referred in US20130287821 and EP2659881.

Examples of useful sequestering agents include disodium ethylene diaminetetraacetate, alkali metal hexametaphosphate, citric acid, sodiumcitrate and mixtures thereof.

Examples of useful viscosity builders include hydroxyethyl cellulose,hydroxymethyl cellulose, HPMC (hydroxypropyl methylcellulose), polyvinylpyrrolidone, polyvinyl alcohol and mixtures thereof.

Examples of useful antioxidants include sodium metabisulfite, sodiumthiosulfate, N-acetylcysteine, butylated hydroxyanisole, butylatedhydroxytoluene, sodium sulfite, potassium sulpfite, sodiummetabisulfite, sodium thiosulfate and mixtures thereof.

The excipients used in the present invention are preferably selected tobe non-toxic and have no substantial detrimental effect (preferably, inthe amount used) on the present ophthalmic solutions, on the use of thesolutions or on the human or animal to which the ophthalmic compositionsare to be administered.

In an embodiment, the present invention provide the ophthalmiccompositions in the form of aqueous liquids, solutions, emulsion,dispersion, suspension, reverse emulsion and microemulsion,nanoemulsion, nano reservoir system, in-situ gel drops, nanoparticulatesystem, liposomal drops, bioadhesive gel drops, drops and the like.

In another embodiment, the present invention preferably provides theophthalmic solution for topical ophthalmic delivery comprisingadministering said solution in the eyes, ear, and/or nose of the humansor animals.

In yet another embodiment, the stable, solution would be an aqueoussolution having a pH value within the range of from about 5.5 to about6.5 and osmolality in range of at least about 200 mOsmol/kg, preferablyin the range of about 200 to about 350 or about 400 mOsmol/kg.

In further embodiment, the present invention provides a process ofpreparing a stable, ophthalmic solution comprising brimonidine tartrateand/or pharmaceutically acceptable excipients, that is more stable thanthe commercial product, Alphagan® P. stability data is shown instability section.

Still further, the present invention may also be presented as a kitcomprising a stable, aqueous solution comprising brimonidine tartrateand/or pharmaceutically acceptable excipients, the aqueous solutionbeing contained within a container prepared from a pharmaceuticallyacceptable packaging material.

Any pharmaceutically acceptable packaging material may be use,preferably packaging material that is suitable for containing ophthalmicaqueous solution, more preferably brimonidine tartrate ophthalmicaqueous solution. Pharmaceutically acceptable packaging materialsinclude but are not limited to low density polyethylene (“LDPE”), highdensity polyethylene (“HDPE”), polypropylene, polystyrene,polycarbonate, polyesters (such as polyethylene terephthalate andpolyethylene naphthalate), nylon, polyvinyl chloride), poly(vinylidinechloride), poly(tetrafluoroethylene) and other materials known to thoseof ordinary skill in the art. Flexible bottles prepared from, orcomprising, LDPE, HDPE or polypropylene are particularly preferred.

The present invention provides a method to lower intraocular pressure inpatients with glaucoma or ocular hypertension wherein the methodcomprises a topical application to the eye of the patient in need of atopical ophthalmic solution comprising an alpha-2 adrenergic receptoragonist such as brimonidine tartrate and/or pharmaceutically acceptableexcipients.

The present invention provides a method of using the inventiveophthalmic solution for lowering intraocular pressure in patients withglaucoma or ocular hypertension.

The present invention provides a process of preparing a topicalophthalmic solution comprising an alpha-2 adrenergic receptor agonistsuch as brimonidine tartrate and/or pharmaceutically acceptableexcipients.

The term preservative used in this invention has the meaning commonlyunderstood in the ophthalmic art. The preservatives comprises one ormore of benzalkonium chloride, benzyldodecinium bromide, chlorobutanol,sodium perborate, cetrimonium chloride, thiomersal, methylparahydroxybenzoate, propyl parahydroxybenzoate, sorbic acid andderivatives thereof, polyquaternium ammonium chloride, polyaminopropylbiguanide, phenyl mercuric nitrate, phenyl mercuric acetate, hydrogenperoxide and the like.

The amount of preservative to be included in the compositions of thepresent invention will generally range from 0.001 to 0.03%, preferably0.001 to 0.015%.

One particularly suitable preservative for use in the compositions ofthe present invention is benzododecinium bromide.

EXAMPLES

The scope of the present invention is illustrated by the followingexample which is not meant to restrict the scope of the invention in anymanner whatsoever.

The term ‘q.s.’ wherever appears in the examples is an abbreviation for‘quantity sufficient’ which is the amount of the excipient in suchquantities that is just sufficient for its use in the composition of thepresent invention.

Example 1

Present Invention Ingredients % w/v Brimonidine Tartrate 0.1 HPMC (E4M)0.5 Boric acid 1.1 Sodium borate 0.07 Sodium chloride 0.18 Potassiumchloride 0.14 Calcium chloride 0.02 Magnesium chloride 0.006Benzododecinium bromide 0.012 Hydrochloric acid q.s. to adjust pH(approx. 6) Sodium hydroxide q.s. to adjust pH (approx. 6) Milli-Q waterq.s. to 1 mL

Example 2

Present Invention Ingredients % w/v Brimonidine Tartrate 0.15 HPMC (E4M)0.5 Boric acid 1.1 Sodium borate 0.07 Sodium chloride 0.18 Potassiumchloride 0.14 Calcium chloride 0.02 Magnesium chloride 0.006Benzododecinium bromide 0.012 Hydrochloric acid q.s. to adjust pH(approx. 6) Sodium hydroxide q.s. to adjust pH (approx. 6) Milli-Q waterq.s. to 1 mL

Manufacturing Process

I. Part A—(Preparation of hydroxypropyl methylcellulose (HPMC) solution)

-   -   1. Add required quantity of HPMC to water for injection (WFI)        (40% of batch size) at 60-70° C. under stirring.    -   2. Cool this solution up to room temperature (up to 25° C.)        under stirring.

II. Part B—(Preparation of API solution)

-   -   1. Take water for injection (50% of batch size) in clean        container.    -   2. Cool water for injection up to room temperature (up to 25°        C.).    -   3. Add and dissolve Boric Acid in above water for injection        under stirring.    -   4. Add and dissolve Sodium Borate to step 3 under stirring.    -   5. Add and dissolve Sodium Chloride to step 4 under stirring.    -   6. Add and dissolve Potassium Chloride to step 5 under stirring.    -   7. Add and dissolve Calcium Chloride to step 6 under stirring.    -   8. Add and dissolve Magnesium Chloride to step 7 under stirring.    -   9. Add and dissolve Benzododecinium Bromide to step 8 under        stirring.    -   10. Add and dissolve Brimonidine Tartrate to step 9 under        stirring.

III. Add Part-B (API solution) to Part-A (HPMC Solution) under stirring.

IV. Check the pH of above solution and adjust the pH of solution with 1Nhydrochloric acid/1N sodium hydroxide solution.

V. Make up the volume up to 100% with water for injection (WFI).

Stability Studies

Stable, topical ophthalmic solutions comprising an alpha-2 adrenergicreceptor agonist such as brimonidine tartrate and/or pharmaceuticallyacceptable excipients are prepared as shown in Table 2 and exposed toaccelerated conditions at 40±2° C. and relative humidity of NMT 25% RHfor 1, 2, 3 and 6 months to determine the stability of the presentinvention as shown in Table 4. An initial study of commerciallyavailable Alphagan® P is initiated to demonstrate the physico-chemicalparameters as shown in Table 3.

TABLE 2 Present Invention Ingredients % w/v Brimonidine Tartrate 0.1HPMC (E4M) 0.5 Boric acid 1.1 Sodium borate 0.07 Sodium chloride 0.18Potassium chloride 0.14 Calcium chloride 0.02 Magnesium chloride 0.006Benzododecinium bromide 0.012 Hydrochloric acid q.s. to adjust pH(approx. 6) Sodium hydroxide q.s. to adjust pH (approx. 6) Milli-Q waterq.s. to 1 mL

Results and Observations

The formulation of Alphagan® P is evaluated to demonstrate thephysico-chemical parameters such as brimonidine tartrate content andrelated substances for accelerated conditions at 40±2° C. and relativehumidity of NMT 25% RH. Results are shown in Table 3.

Initial Testing of Commercial Product

TABLE 3 Commercial product Commercial product Parameters (Alphagan ® P)(Alphagan ® P) Description Clear, greenish Clear, greenish yellowcolored yellow colored solution solution PH 7.60 7.60 Osmolality 265mOsmol/Kg 266 mOsmol/Kg Viscosity 3.05 cps 3.13 cps Assay of Brimonidine96.2% 96.2% Tartrate Related Substances a Debromobrimonidine — — b6-Amino-quinoxalina ND ND c 5-Bromoquinoxaline-6- ND ND Amine d HighestUnknown 1.49% 1.43% Impurity e Other Unknown Impurity 1.16% 1.43% fTotal Impurities 2.65% 2.86%

The ophthalmic solution of present invention is evaluated to demonstratethe physico-chemical parameters such as brimonidine tartrate content andrelated substances at 1, 2, 3 and 6 months for accelerated conditions at40±2° C. and relative humidity of NMT 25% RH. Results are shown in Table4.

TABLE 4 Stability Data of Brimonidine Tartrate Ophthalmic Solution ofthe present invention: Batch No. PR3F044-06 40 ± 2° C./NMT 25% RHParameters Initial 1 Month 2 Month 3 Month 6 Month Description Clear,Clear, Clear, Clear, Clear, greenish slightly slightly slightly slightlyyellow greenish greenish greenish greenish colored yellow yellow yellowyellow solution colored colored colored colored solution solutionsolution solution pH 6.04 5.92 5.99 5.92 5.56 Osmolality 283 281 279 293302 mOsmol/Kg mOsmol/Kg mOsmol/Kg mOsmol/Kg mOsmol/Kg Viscosity 17.5 cps17.0 cps 16.2 cps 15.7 cps 14.7 cps Assay of Brimonidine 96.0% 99.7%99.8% 99.3% 101.0%  Tartrate Content of 91.0% 88.8% 92.3% 90.3% 87.7%Benzododecinium Bromide Related Substances a Debromobrimonidine ND 0.01%0.01% 0.01% 0.29% b 6-Amino-quinoxalina ND ND ND ND ND c 5- ND 0.02%0.02% 0.04% 0.10% Bromoquinoxaline- 6-Amine d Highest Unknown 0.03%0.03% 0.05% 0.09% 0.014%  Impurity e Other Unknown 0.03% ND 0.04% 0.11%0.12% Impurity f Total Impurities 0.03% 0.06% 0.12% 0.25% 0.65%

The Results at Accelerated Conditions at 40±2° C. and Relative Humidityof NMT 25% RH for Alphagan® P as a Commercial Product:

The brimonidine tartrate content is measured and found to be 96.2%(Limit: 90.0-110.0%) which is within the acceptable limit range, thehighest unknown impurity is measured and found to be 1.49% (Limit: NMT1.0%) which is above the acceptable limit range and total impurity ismeasured and found to be 2.65% (Limit: NMT 3.0%) which is within theacceptable limit range.

The Results at Accelerated Conditions at 40±2° C. and Relative Humidityof NMT 25% RH for 6 Months for the Present Invention:

The brimonidine tartrate content is measured and found to be 101%(Limit: 90.0-110.0%) which is within the acceptable limit range, thehighest unknown impurity is measured and found to be 0.014% (Limit: NMT1.0%) which is within the acceptable limit range and total impurity ismeasured and found to be 0.65% (Limit: NMT 3.0%) which is within theacceptable limit range.

The above stability study of the present invention indicates that theall the parameters of the present invention are well within narrowlimits and the solution prepared by the present invention formulation isstable with respect to all physico-chemical parameters which indicatethat at stability testing conditions there appears to be no considerabledegradation in the present solution.

Comparison of Impurity Profile of Commercial Product (Alphagan® P) andPresent Invention Formulation (Brimonidine Tartarate 0.1%):

Further the two (2) lots of Commercial product {Alphagan® P (0.1%)} andfour (4) lots of present invention formulation (brimonidine tartarate0.1%) are compared at accelerated stability conditions (40° C./NMT 25%RH) and then compared the Impurity profiles of the two and the resultsare tabulated in Tables 5 and Table 6.

TABLE 5 (Commercial product {Alphagan ® P (0.1%)}) Commercial product{Alphagan ® P (0.1%)} Related Lot No-79087 Lot No-77611substances/Impurity Initial 1 M 2 M 3 M 6 M Initial 1 M 2 M 3 M 6 MDebromobrimonidine 0.01 0.01 0.02 0.02 0.03 0.01 0.01 0.02 0.02 0.026-Amino- quinoxaline ND ND ND 0.01 ND ND ND ND ND ND5-Bromoquinoxaline-6-amine 0.03 0.08 0.11 0.17 0.35 0.03 0.06 0.13 0.240.52 Highest Unknown Impurity 0.30 0.60 0.86 1.18 1.85 0.26 0.58 0.911.33 1.12 Unknown Impurity 0.58 0.62 0.63 0.6 1.06 0.49 0.64 0.92 0.962.17 Total Impurity 0.92 1.31 1.62 1.98 3.29 0.79 1.29 1.98 2.55 3.83

TABLE 6 {Present invention formulation (brimonidine tartarate 0.1%)}{Present invention formulation (brimonidine tartarate 0.1%)} RelatedPR3F044-06 PR3F044-22 substances/Impurity Initial 1 M 2 M 3 M 6 MInitial 1 M 2 M 3 M 6 M Debromobrimonidine *ND  0.01 0.01 0.01 0.29 BQLBQL 0.02 0.15 0.04 6-Amino-quinoxaline ND ND ND ND ND ND ND ND BQL 0.015-Bromoquinoxaline-6-amine ND 0.02 0.02 0.04 0.1  ND 0.01 0.02 0.05 NDHighest Unknown Impurity 0.03 0.03 0.05 0.09 0.14 BDL BDL 0.09 0.13 0.49Unknown Impurity 0.03 ND 0.04 0.11 0.12 ND BDL 0.15 0.11 0.66 TotalImpurity 0.03 0.06 0.12 0.25 0.65 BDL 0.01 0.28 0.44 1.2 {Presentinvention formulation (brimonidine tartarate 0.1%)} Related PR3F044-31PR3F044-32 substances/Impurity Initial 1 M 2 M 3 M 6 M Initial 1 M 2 M 3M 6 M Debromobrimonidine **BQL 0.02 0.03 0.04 Ongoing BQL 0.02 0.04 0.05Ongoing 6-Amino-quinoxaline ND BQL ND BQL ND BQL ND BQL5-Bromoquinoxaline-6-amine ND ND 0.01 0.01 ND 0.01 0.01 0.02 HighestUnknown Impurity ***BDL   0.06 0.1 0.14 BDL 0.06 0.1 0.14 UnknownImpurity BDL ND BDL BDL BDL ND BDL BDL Total Impurity BDL 0.08 0.14 0.19BDL 0.09 0.15 0.21 *ND—Not Detected **BQL—Below Quantitation Limit***BDL—Below Disregard Limit.

The total Impurity levels in various brimonidine formulations i.e.Commercial product (Alphagan® P) and present invention formulation(brimonidine tartarate 0.1%) at 40° C./NMT 25% RH are tabulated in Table7 and as plotted in FIG. III:

TABLE 7 Total Impurity Level (%) Commercial product (Alphagan ® P)Present invention formulations Time Alphagan ® Alphagan ® PresentPresent Present Present (mon ths) P 79087 P 77611 Invention-06Invention-22 Invention-31 Invention-32 0 0.92 0.79 0.03 BDL BDL BDL 11.31 1.29 0.06 0.01 0.08 0.09 2 1.62 1.98 0.12 0.28 0.14 0.15 3 1.982.55 0.25 0.44 0.19 0.21 6 3.29 3.83 0.65 1.2ANIMAL EFFICACY STUDIES: Two studies were conducted to compare theefficacy of the present invention formulation with the commercialproduct (Alphagan® P) in two different models:

-   1. Normotensive model-   2. Water loaded model.

1. NORMOTENSIVE MODEL Objective

The objective of the study is to test and compare the IOP loweringefficacy of two formulations (Present Invention and commercial product)on normotensive New Zealand white rabbits.

Materials and Methods

Twelve adult female New Zealand white rabbits are selected for thisstudy. Body weights of all 12 rabbits are within ±20% of the mean bodyweight at the time of randomization. The selected animals are randomlygrouped into two groups having 6 animals per group. Group I is treatedwith Alphagan® P and Group II is treated with another formulation(Present Invention), topically in left eye. The other (right) eye isleft untreated and serves as control.

Dose Level

50 μL of each formulation was instilled once in left eye.

Test

After randomization and grouping, intra ocular pressure is measuredinitially for 24 hours at 2 hours interval to observe the degree ofdiurnal variations. On day 1 of experiment, baseline IOP is measured forboth eyes of each animal. After baseline estimation, the commercialformulation (SRC/Aravali/78115) is instilled in left eye of all rabbitsin group I and another present invention formulation (SRC/Aravali/022)in group II. The contra lateral eye serves as control.

Subsequent to the test item instillation, IOP estimations are repeatedat 1 hr intervals until the baseline IOP is achieved.

OBSERVATIONS Cageside Observations & Physical Examinations

All the animals are observed for cageside observations and physicalexaminations for 2 days for each group.

Mortality

All the animals are observed for mortality for 2 days of experiment.

Intra Ocular Pressure Measurement

After randomization and grouping, and before initiating the experiment,intra ocular pressure is measured initially for 24 hours at 2 hoursinterval to observe the degree of diurnal variations. On the testing daybefore the instillation of test item IOP is measured to determine thebaseline IOP and after every 1 hour interval after dosing till thebaseline IOP is achieved.

INTERPRETATION OF RESULTS

IOP readings of formulations instilled in the left eye of each animalare compared for IOP lowering efficacy with respect to the extent andduration within its own group. At the same time IOP lowering ability ofboth formulations is compared between the groups of animals i.e.commercial formulation (SRC/Aravali/78115) and present inventionformulation (SRC/Aravali/022). To derive the statistical significance,Student's t test is used for IOP data. The statistical significance isdisclosed at 95% confidence interval (p<0.05).

RESULTS AND DISCUSSION Cageside Observations & Physical Examinations

All the animals are normal in appearance throughout the observationperiod. The cageside observations and physical examinations data arereported in Table 8.

TABLE 8 Cage side observations & physical examinations Cage sideobservations Group No. of animals Clinical & physical examination No.used sign Day 0 Day 1 I 6F Normal 6 of 6 6 of 6 II 6F Normal 6 of 6 6 of6

Mortality

There is no mortality observed in both dose groups. The mortality datais reported in

Table 9.

TABLE 9 Mortality No. of animals Mortality Group No. used Day 0 Day 1 I6F 0/6 0/6 II 6F 0/6 0/6

Intra Ocular Pressure Measurement

Diurnal intra ocular pressure measured for 24 hr (at 2 hr interval) doesnot have any statistically significant (p<0.05) difference among leftand right eyes of animals within the group for both groups. Intraocularpressure for left eye of Groups I and II are also comparable and this isalso true for right eye of both groups.

Before test item instillation baseline IOP is measured and then for thenext 5 hrs at 1 hr intervals. Peak statistically significant (p<0.05)drop of IOP in Groups I & II is observed at 2nd hour of instillation ascompare to control eye. When particular time point is compared withbaseline statistically significant difference is observed at 2nd and 3rdhour in both groups.

At the same time, IOP readings of animals from Group I are compared withanimals of Group II and there are no statistically significant changesobserved. The extent and duration of IOP lowering effect of twoformulations {commercial product (Alphagan® P) and present invention}are comparable.

Based on observations obtained from the present study, it is concludedthat two formulations {commercial product (Alphagan® P) and presentinvention} have statistically significant IOP lowering effect.

The intra ocular pressure data for Normotensive Model is reported inTable 10 and presented in FIG. I.

TABLE 10 Intra ocular pressure (mmHg) Baseline & after instillationBase- Group Eye line 1 hr 2 hr 3 hr 4 hr 5 hr I Left Mean 21.0 20.1 14.516.4 19.0 20.3 SEM 0.9 0.7 0.7 0.6 0.7 0.6 P VALUE — 0.56 0.00 0.00 0.110.55 (BL Vs. time) Right Mean 20.3 19.8 19.8 18.2 20.6 20.9 SEM 0.3 0.71.0 0.9 0.5 0.5 P VALUE — 0.51 0.63 0.05 0.59 0.35 (BL Vs. time) II LeftMean 20.0 19.2 15.7 16.0 19.3 20.9 SEM 0.4 0.5 0.6 0.9 0.8 0.5 P VALUE —0.21 0.00 0.00 0.37 0.20 (BL Vs. time) Right Mean 18.9 17.9 19.1 17.420.3 19.8 SEM 0.6 0.7 1.0 0.8 0.3 0.4 P VALUE — 0.27 0.92 0.15 0.06 0.28(BL Vs. time) N = 6 animals per group; SEM = Standard Error of Mean; BL= Baseline.

CONCLUSION

Based on the observations obtained from this study, it is concluded thatthe two formulations, {commercial product (Alphagan® P) and presentinvention} show statistically significant IOP lowering effect whenadministered to New Zealand white rabbits and the efficacy of both theformulations is statistically comparable.

2. WATER LOADED MODEL Objective

The objective of the study is to test and compare the IOP loweringefficacy of two different formulations on water loaded New Zealand whiterabbits.

Materials and Methods

Twelve adult female New Zealand white rabbits those used in normotensivestudy are used for this study. The animals are randomly grouped into twodifferent groups containing 6 animals per group at the time ofrandomization in normotensive study. Group I is treated with commercialproduct (Alphagan® P) and Group II is treated with another presentinvention formulation, topically in left eye. Same grouping is followedfor both the dose groups as followed in normotensive study.

Dose Level

50 μL of each formulation is instilled once in left eye.

Test

After weighing, the baseline IOP is noted and rabbits are first orallyadministered with water @70 mL/kg through orogastric tube and IOP ismeasured till the baseline IOP achieved (for 2.5 hrs) at 0.25 hrinterval to observe the IOP pattern. The time at which the peak IOP isachieved is noted.

On the next day of experiment, initially baseline IOP is measured forboth eyes. After the baseline estimation, commercial product (Alphagan®P) formulation is instilled in left eye of all rabbits in Group I andpresent invention in Group II. The right eye serves as control.

Test item instillation is followed by rapid oral administration of water(70 mL/kg) through an orogastric tube.

The interval between drug administration and water loading is kept at1.5 hr based on the following observations, time to peak intraocularpressure lowering effect of the drug as observed in normotensive rabbitstudy numbered 132101 (2 hrs) and the time of peak IOP elevationobserved in water loaded untreated rabbits (15 min to 90 min) in thisstudy numbered 132102. Afterwards, IOP estimations are carried out for2.25 hours at 0.25 hr interval till the IOP returns to original baselineIOP.

OBSERVATIONS Cageside Observations & Physical Examinations

All the animals are observed for cageside observations & physicalexaminations for 2 days for each group.

Mortality

All the animals are observed for mortality for 2 days of experiment.

Intra Ocular Pressure Measurement

After weighing of animals, the baseline IOP is measured and then rabbitsare orally administered with water @70 mL/kg through orogastric tube andIOP is measured till the baseline IOP achieved (for 2.5 hrs) at 0.25 hrinterval to observe the IOP pattern. The time at which the peak IOPachieved is noted.

On the day of test item instillation, IOP is measured prior to the testitem instillation for baseline IOP estimation. After baseline IOPmeasurement, test items are instilled to respective group, which isfollowed by rapid oral water loading. IOP estimations are measured atevery 0.25 hour interval after water loading till the baseline IOP isachieved.

INTERPRETATION OF RESULTS

IOP readings of the test item instilled in the left eye of each animalare compared for IOP lowering effect with respect to the extent andduration within its own group. At the same time IOP lowering ability ofboth test items is compared in two groups of animal's i.e. commercialproduct (Alphagan® P) formulation and present invention groups. Toderive the statistical significance, Student's t test is used for IOPdata. The statistical significance is disclosed at 95% confidenceinterval (p<0.05).

RESULTS AND DISCUSSION Cageside Observations & Physical Examinations

All the animals are normal in appearance throughout the observationperiod. The cageside observations and physical examinations data arereported in Table 11.

TABLE 11 Cage side observations & physical examinations Cage sideobservations & physical examination No. of Day of animals Day of waterinstillation & Group No. used Clinical sign leading only water loading I6F Normal 6 of 6 6 of 6 II 6F Normal 6 of 6 6 of 6

Mortality

There is no mortality observed in both dose groups. The mortality dataare reported in Table 12.

TABLE 12 Mortality Mortality No. of animals Day of water Day ofinstillation Group No. used loading only & water loading I 6F 0/6 0/6 II6F 0/6 0/6

Intra Ocular Pressure Measurement

On first day, initially baseline IOP is measured and then rabbits areadministered with water @70 mL/kg through orogastric tube and IOP ismeasured till the baseline IOP achieved (for 2.5 hrs) at 0.25 hrinterval to observe the IOP pattern. Statistically significant (p<0.05)increased intraocular pressure is observed from 15 min to 90 min in botheyes of rabbits from Group I, from 15 min to 75 min in left eye and 15min to 90 min in right eye of rabbits from Group II as compared to itsbaseline reading. At the same time, when within the group left eyes arecompared with right eyes, it shows comparable increase in IOP and nostatistically significant (p<0.05) changes in IOP is observed for bothdose groups. Same way, when left and right eyes of Group I are comparedwith left and right eyes of Group II, there are no statisticallysignificant (p<0.05) changes observed in IOP, except significant higherIOP is observed in left eye of Group I at baseline and 15 minutes ascompare to left eye of Group II, which is believed attributable tobiological variation.

On the next day, test items are instilled in left eye of respectivegroups after baseline IOP measurement. 1.5 hours after test iteminstillation, animals are orally dosed rapidly with 70 mL/kg of water.This time gap is derived from time to peak intraocular pressure loweringeffect of the drug observed in normotensive rabbit study numbered 132101which was 2 hr and the time of peak IOP elevation observed in waterloaded untreated animals (15 min-90 min) in this study numbered 132102.

As compare to baseline, there is a statistically significant (p<0.05)increase in IOP at 15 min in left eyes of both dose groups, at 15 to 75min in right eye of Group I and at 15 to 105 min in right eye of GroupII. In left eyes (treated eyes) of both dose groups, from 30 min onwardIOP is reduced and becomes statistically non-significant (p<0.05) ascompared to baseline.

At the same time, when both left and right eyes are compared, there isstatistically significant (p<0.05) lower IOP observed in left eyes ascompared to right eyes from 30 min to 75 min in Group I and from 15 minto 105 min in Group II.

Same way, when left and right eyes of Group I are compared with left andright eyes of Group II, there are no statistically significant (p<0.05)changes observed in IOP, except significant (p<0.05) slight higher IOPobserved at 90 minute in left eyes of Group I as compared to left eyesof Group II. This difference might be due to difference in baseline ofleft eyes of both groups and considered as biological variation.

Lower IOP levels in left eyes of rabbits belonging to both dose groupsindicate desired IOP lowering efficacy of both test items. Also, IOPlevels in left eyes of both dose groups are comparable hence it can beconcluded that the effect of both test items are equivalent to eachother. The extent and duration of present invention is longer by 0.5hour than commercial product (Alphagan® P) formulation as Group IIreveals IOP lowering effect up to 105 min as compare to Group I, whichreveals IOP lowering effect up to 75 min and response to presentinvention started 15 min earlier as compare to commercial product(Alphagan® P) formulation when compared to right eyes of respectivegroups.

Based on observations obtained from present study, it is concluded thattwo formulations {commercial product (Alphagan® P) formulation andpresent invention} have statistically significant IOP lowering efficacyand are comparable to each other. Present invention reveals slightlylonger duration of action than the commercial product (Alphagan® P)formulation, when compared to right eyes of respective groups.

The intra ocular pressure data for Water Loaded Model are reported inTable 13 and presented in FIG. II.

TABLE 13 Intra ocular pressure (mmHg) Baseline & after instillation12:45 13:00 13:15 13:30 13:45 14:00 14:15 14:30 14:45 9:30 15 30 45 6075 90 105 120 135 Group Eye Baseline min min min min min min min min minI Left Mean 19.8  24.9 21.3 20.7 18.2 18.7 19.8 19.5 19.5 18.9 SEM 0.41.3 1.1 0.8 0.8 0.7 0.6 0.6 0.6 0.0 P VALUE — 0.00 0.21 0.31 0.12 0.190.98 0.70 0.70 0.05 (BL Vs. time) Right Mean 19.2  27.5 27.5 27.8 25.123.7 21.2 20.8 20.7 20.4 SEM 0.7 2.2 2.3 2.0 1.8 1.8 1.6 1.2 0.9 0.7 PVALUE — 0.00 0.01 0.00 0.01 0.04 0.29 0.30 0.22 0.26 (BL Vs. time) IILeft Mean 18.9  21.6 19.0 19.2 17.4 17.9 17.4 17.9 18.9 19.2 SEM 0.4 0.90.9 0.5 0.6 0.7 0.5 0.5 0.4 0.5 P VALUE — 0.02 0.91 0.68 0.07 0.22 0.050.14 1.00 0.68 (BL Vs. time) Right Mean 19.2  26.1 25.5 25.5 24.9 23.222.2 20.9 20.7 20.3 SEM 0.3 1.5 2.1 2.0 1.0 1.0 0.9 0.7 0.8 0.5 P VALUE0.00 0.01 0.01 0.00 0.00 0.01 0.04 0.12 0.09 (BL Vs. time) N = 6 animalsper group; SEM = Standard Error of Mean; BL = Baseline.

CONCLUSION

Based on the observations obtained from this study, it is concluded thatthe two formulations, commercial product (Alphagan® P) formulation andthe present invention, demonstrate statistically significant IOPlowering efficacy when administered to water loaded New Zealand whiterabbits by ocular route. The onset of statistically significant IOPlowering efficacy is 15 minutes earlier in present invention as comparedto commercial product (Alphagan® P) formulation, whereas both theformulations are found to be comparable at each time point observed,which indicates that the IOP lowering efficacy of both test formulations(commercial product (Alphagan® P) formulation and present invention) arestatistically comparable.

1. A method of reducing elevated intraocular pressure in a patient inneed thereof, the method comprising administering a therapeuticallyeffective amount of a composition comprising: 0.05-0.2% (w/v) ofbrimonidine tartrate; and about 0.1-1.0% (w/v) hydroxypropyl methylcellulose; wherein the composition is administered to at least one eyeof the patient.
 2. (canceled)
 3. The method of claim 1, wherein thecomposition comprises 0.15% (w/v) of brimonidine tartrate.
 4. The methodof claim 1, wherein the composition comprises 0.1% (w/v) of brimonidinetartrate. 5-8. (canceled)
 9. The method of claim 1, wherein thecomposition is highly soluble at a pH of 6.5 or less.
 10. The method ofclaim 1, wherein the pH of the composition is in the range of 5.5-6.5.11. The method of claim 1, wherein the composition further comprises aquaternary ammonium compound as a preservative in the amount of0.001-1.0% (w/v).
 12. (canceled)
 13. The method of claim 11, wherein thesole quaternary ammonium compound included in the composition idbenzododecinium bromide.
 14. The method of claim 1, wherein thecomposition is further devoid of an oxidative preservative.
 15. A methodof lowering the intraocular pressure in a patient with glaucoma orocular hypertension, comprising administering to the patient atherapeutically effective amount of a composition comprising: 0.05-0.2%(w/v) of brimonidine tartrate; and about 0.1-1.0% (w/v) hydroxypropylmethyl cellulose. 16-24. (canceled)
 25. The method of claim 1, whereinthe composition is devoid of an anionic solubility enhancing component.26. (canceled)
 27. The method of claim 1, wherein the compositioncomprises: a) 0.1% (w/v) brimonidine tartrate; b) 0.5% (w/v)hydroxypropyl methylcellulose; and c) 0.012% (w/v) benzododecinium, andpharmaceutically acceptable excipients, wherein the composition ishighly soluble at a pH of 6.5 or less. 28-30. (canceled)